: Pruritus, also called itch, is the most frequent dermatological symptom in the US with up to 20% of Americans being affected. Pruritic stimuli are detected by a subset of nociceptive neurons, pruriceptors, which fire action potential in response to peripheral activation by TH2 or TH17 molecules induced by chronic inflammatory skin diseases such as ichthyosis, prurigo nodularis, and hidradenitis suppurativa. T-type calcium channels contribute to the firing behavior of itch-sensing neurons in synaptic skin nerve terminals and have recently been discovered to mediate calcium entry in immune cells. This T-type calcium channel expression pattern is consistent with their critical roles at detecting itch sensations associated with skin inflammatory diseases. Preliminary work has enabled us to formulate the central hypothesis that topical inhibition of the three T-type channel isoforms in the skin will silence action potential (AP) discharge of itch A? and C-fibers induced by pruritogenic and inflammatory cytokines, will directly reduce the release of these cytokines by TH2 or TH17 cells, and will lead to improved efficacy and limited systemic exposure, together reducing the risk of adverse effects compared to systemic treatments. Our hypothesis is strongly supported by our experimental findings. First, our lead compound, DX416, is a potent inhibitor of the three T-type channels, Cav3.1, Cav3.2, and Cav3.3 at sub-micromolar levels. Second, treatment with DX416 does not induce psychoactivity in rodents but profoundly reduces histamine-evoked itch in mice after intradermal administration. Third, we discovered DX401, a T-type blocker that reduces histamine-evoked itch after oral administration. Fourth, we show for the first time that these two Cav3s blockers, DX416 and DX401, significantly reduce the release of TH2 pruritic cytokine, IL- 31, after human peripheral blood mononuclear cell (PBMC) activation, and TH17-synergizing cytokine, TNF?, by activated macrophages. The overall goal of this collaborative effort between DermaXon?s experts in medicinal chemistry, immunology, and dermatology is to demonstrate the technical feasibility of developing inhibitors of skin T-type channels as novel therapeutics for the topical treatment of pruritus associated with inflammatory skin diseases. Our objectives in this proposal are: Aim 1) complete the optimization of our first topical T-type lead inhibitor DX416, based on our previously designed chemotypes and characterize their Cav3s activity and selectivity; Aim 2) compare the effect of four Cav3s blockers selected in SA1 on proinflammatory and pruritogenic cytokine release by activated immune cells, and assess their safety; Aim 3) compare the anti-pruritic efficacy after intra-dermal administration of two T-type channels inhibitors selected in Aim 2 using the histamine- evoked acute itch and the dry skin chronic itch in vivo models. The identification of lead compounds that are active in preclinical acute and chronic itch models will set the stage for a next generation of topical pruritus therapeutics to be used in both adult and pediatric patients suffering from itch associated with inflammatory skin diseases, and will support development of a novel therapeutic topical treatment.